The adhesion of mammalian cells to the extracellular matrix is of fundamental importance in regulating growth, adhesion, motility and the development of the proper cellular phenotype. This has implications for normal cell growth and development, wound healing, chronic inflammatory diseases, diabetes, and tumor metastasis. Evidence accumulated over the last several years suggests that the molecular basis for the adhesion of both normal and transformed or malignant cells is complex and probably involves several distinct cell surface molecules. Extracellular matrices consist of three types of macromolecules: collagenous glycoproteins, proteoglycans and noncollagenous glycoproteins.
One noncollagenous adhesive glycoprotein of interest is laminin. Laminin likely is one of a multigene family of related molecules [Ohno et al., Conn. Tiss. Res., 15, 199-207 (1986); Hunter et al., Nature, 338, 229-234 (1989); Sanes et al., J. Cell Biol., 111, 1685-1699 (1990)]. Laminin is a high molecular weight (approximately 850,000; from the mouse Engelbreth-Holm-Swarm tumor) extracellular matrix glycoprotein found almost exclusively in basement membranes. [Timpl et al., J. Biol. Chem., 254, 9933-9937 (1979)]. Basement membranes are an ubiquitous, specialized type of extracellular matrix separating organ parenchymal cells from interstitial collagenous stroma. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes. Normal cells appear to require an extracellular matrix for survival, proliferation, and differentiation, while migratory cells, both normal and neoplastic, must traverse the basement membrane in moving from one tissue to another.
Laminin isolated from the Engelbreth-Holm-Swarm murine tumor consists of three different polypeptide chains: B1 with 215,000 MW, B2 with 205,000 MW and A with 400,000 MW [Timpl and Dziadek, Intern. Rev. Exp. Path., 29, 1-112 (1986)]. When examined at the electron microscopic level with the technique of rotary shadowing, it appears as an asymmetric cross, with three short arms 37 nm long (the lateral short arms having two globular domains and the upper short arm having three globular domains [Sasaki et al., J. Biol. Chem., 263, 16536-16544 (1988)]}, and one long arm 77 nm long, exhibiting a large terminal globular domain [Engel et al., J. Mol. Biol., 150, 97-120 (1981)]. The three chains are associated via disulfide and other chemical bonds. Structural data shows that laminin is a very complex and multidomain protein with unique functions present in specific domains.
Laminin is a major component of basement membranes and is involved in many functions. Laminin has the ability to bind to other basement membrane macromolecules and therefore contributes to the structural and perhaps functional characteristics of basement membranes.
Laminin promotes the adhesion and spreading of a multitude of cells and binds a variety of proteoglycans [Timpl and Dziadek, supra (1986)]. Studies utilizing enzymatic digests of laminin or monoclonal antibodies raised against laminin have defined some of the biologically active regions of the 400 kD A chain of laminin. The amino terminal globular domain at the top of the molecule is involved in laminin-laminin self assembly [Yurchenco et al., J. Biol. Chem., 260, 7636-7644 (1985)] and the adhesion of hepatocytes [Timpl et al., J. Biol. Chem., 255, 8922-8927 (1983)]. In addition, its large carboxy-terminal globular domain binds heparin [Ott et al., Eur. J. Biochem., 123, 63-72 (1982); Skubitz et al., J. Biol. Chem., 263, 4861-4868 (1988)], while neurite cell outgrowth and cell adhesion is localized to the region directly above this globule [Edgar et al., EMBO J., 3, 1463-1468 (1984); Engvall et al., J. Cell Biol., 103, 2457-2465 (1986); Goodman et al., J. Cell Biol., 105, 589-598 (1987)].
Another important feature of laminin is its ability to associate with cell surface molecular receptors and consequently modify cellular phenotype in various ways. Receptors for laminin ranging in molecular size from 55 to 180 kD have been isolated from a variety of normal and malignant cell lines [Rao et al., Biochem. Biophys. Res. Commun., 111, 804-808 (1983); Lesot et al., EMBO J., 2, 861-865 (1983); Malinoff and Wicha, J. Cell Biol., 96, 1475-1479 (1983); Terranova et al., Proc. Natl. Acad. Sci. U.S.A., 80, 444-448 (1983); Barsky et al., Breast Cancer Res. Treat., 4, 181-188 (1984); von der Mark and Kuhl, Biochim. Biophys. Acta., 823, 147-160 (1985); Wewer et al., Proc. Natl. Acad. Sci. U.S.A., 83, 7137-7141 (1986); Hinek et al., J. Cell Biol., 105, 138a (1987); Yoon et al., J. Immunol., 138, 259-265 (1987); Smalheiser and Schwartz, Proc. Natl. Acad. Sci. U.S.A., 84, 6457-6461 (1987); Yannariello-Brown et al., J. Cell Biol., 106, 1773-1786 (1988); Mercurio and Shaw, J. Cell Biol., 107, 1873-1880 (1988); Kleinman et al., Proc. Natl. Acad. Sci. U.S.A., 85, 1282-1286 (1988); Hall et al., J. Cell Biol., 107, 687-697 (1988); Clegg et al., J. Cell Biol., 107, 699-705 (1988)]. In the case of human glioblastoma cells [Gehlsen et al., Science, 241, 1228-1229 (1988)], several chicken cell types [Horwitz et al., J. Cell Biol., 101, 2134-2144 (1985)], platelets [Sonnenberg et al., Nature, 336, 487-489 (1988)], and neuronal cell line PC12 [Tomaselli et al., J. Cell Biol., 105, 2347-2358 (1987); and J. Cell Biol., 107, 1241-1252 (1988)], the laminin receptor was determined to be an integrin. To date, however, the exact sequence of amino acid residues of laminin to which most of these receptors bind is unknown.
Recently, the amino acid sequences of the B1, B2, and A chains of laminin have been determined [Barlow et al., EMBO J., 3, 2355-2362 (1984); Sasaki et al., Proc. Natl. Acad. Sci. U.S.A., 84, 935-939 (1987); Sasaki et al., J. Biol. Chem., 263, 16536-16544 (1988); Sasaki and Yamada, J. Biol. Chem., 262, 17111-17117 (1987); Pikkarainen et al., J. Biol. Chem., 262, 10454-10462 (1987); Pikkarainen et al., J. Biol. Chem., 263, 6751-6758 (1988); Hartl et al., Eur. J. Biochem., 173, 629-635 (1988)], allowing peptides to be synthesized from domains of laminin with reported functional activity. Three peptides have been synthesized from the B1 chain of laminin which promote cell adhesion. The first peptide, cys-asp-pro-gly-tyr-iso-gly-ser-arg (SEQ ID NO: 6), located near the intersection of the cross, promotes the adhesion of a variety of cells [Graf et al., Cell, 48, 989-996 (1987a); Biochemistry, 26, 6896-6900 (1987b)] and is thought to bind a 67 kD protein laminin receptor [Graf et al., supra (1987b); Wewer et al., supra (1986)]. The second, peptide F-9 (arg-tyr-val-val-leu-pro-arg-pro-val-cys-phe-glu-lys-gly-met-asn-tyr-thr-v al-arg) (SEQ. ID. NO: 7), also promotes the adhesion of a variety of cells and binds heparin (U.S. Pat. No. 4,870,160) Charonis et al. J. Cell Biol., 107, 1253-1260 (1988). A third peptide from the B1 chain of laminin, termed AC15 {arg-ile-gln-asn-leu-leu-lys-ile-thr-asn-leu-arg-ile-lys-phe-val-lys (SEQ. ID. NO: 8) [Kobuzi-Koliakos et al., J. Biol. Chem., 264, 17971-17978 (1989)]} also binds heparin, and is derived from the outer globule of a lateral short arm. AC15 promotes the adhesion of murine melanoma and bovine aortic endothelial cells [Koliakos et al., J. Cell Biol., 109, 200a (1989)].
Since the A chain was the last chain of laminin for which the entire amino acid sequence was determined, only a few peptides have been described with functional activity. Synthetic peptide PA 21 (residues #1115-1129; cys-gln-ala-gly-thr-phe-ala-leu-arg-gly-asp-asn-pro-gln-gly) (SEQ. ID. NO: 9), which contains the active sequence RGD, induces the attachment of human endothelial cells through an integrin receptor [Grant et al., Cell, 58, 933-943 (1989)]. Peptide PA22-2 (residues #2091-2108; ser-arg-ala-arg-lys-gln-ala-ala-ser-ile-lys-val-ala-val-ser-ala-asp-arg), (SEQ. ID. NO: 10), contains the active sequence ile-lys-val-ala-val (SEQ. ID. NO: 11) [Tashiro et al., J. Biol. Chem., 264, 16174-16182 (1989)] and has a number of biological functions such as promoting neuronal process extension [Tashiro et al., supra (1989); Sephel et al., Biochem. Biophys. Res. Commun., 162, 821-829 (1989)]. Recently the biological activity of this peptide has come into question.
The functions that have been described above make laminin an important component of many diverse and clinically important processes such as cell migration, cell adhesion, cell growth, cell differentiation, wound healing, angiogenesis in general, nerve regeneration, tumor cell invasion and metastasis [Liotta, Am. J. Path., 117, 339-348 (1984); McCarthy et al., Cancer Met. Rev., 4, 125-152 (1985)], diabetic microangiopathy, and vascular hypertrophy due to hypertension, atherosclerosis and coronary artery disease, and vessel wall healing after angioplasty. Laminin could also be used in various devices and materials used in humans. In order to better understand the pathophysiology of these processes at the molecular level, it is important to try to assign each of the biological activities that laminin exhibits to a specific subdomain or oligopeptide of laminin. If this can be achieved, potentially important pharmaceuticals based on small peptides or compounds that bind to the receptors producing specific functions of the native, intact molecule, can be synthesized.
Therefore, a need exists to isolate and characterize peptides which are responsible for the wide range of biological activities associated with laminin. Such lower molecular weight oligopeptides would be expected to be more readily obtainable and to exhibit a narrower profile of biological activity than laminin itself, thus increasing their potential usefulness as therapeutic or diagnostic agents.